Verapamil, a Ca(2+) channel blocker widely used in clinical practice, also affects the properties of frog and mouse muscle acetylcholine receptor (AChR). Here, we examine the mechanism of action of verapamil on human wild-type and slow-channel mutant muscle AChRs harboring in any subunit a valine-to-alanine mutation of 13' residue of the pore-lining M2 transmembrane segment. Verapamil, after a pre-treatment of 0.5-10 s, accelerated the decay of whole-cell or macroscopic outside-out currents within milliseconds of ACh application even at clinically attainable doses. Recordings of unitary events in the cell-attached and outside-out configurations showed that verapamil does not alter single-channel conductance, but reduces channel open probability, by prolonging the dwell time into the closed state for wild-type and all mutant AChR. The duration of channel openings decreased only for the epsilonV265A-AChR, by shortening the longest exponential component of the open-time distribution. These results provide a rationale for the therapeutic use of verapamil in the slow-channel syndrome and emphasize the major role played by epsilon subunit in controlling the functional properties of human muscle AChR, as revealed by the peculiar alterations imparted by mutations in this subunit.